In this invention, an immunodeficient rodent is modified in order that when it receives a donor organ, or portion thereof, or a foreign agent, such as an antigen, the modified rodent mounts a T cell mediated response to the foreign organ or agent in a manner which permits its use as a model for human immune response to the organ or agent. One of the key aspects of this invention is that the rodent receives an organ graft combining microvessels lined with human endothelial cells as opposed to human T cells alone, enabling the rodent to mount a T cell mediated response which corresponds to the interaction of human T cells and human endothelial cells. In many cases, primates have been required to study such T cell mediated responses. Because of costs and difficulties associated with primate models, the various embodiments of this invention should enable significant advancement in understanding human immune responses and developing therapeutics relating thereto.
As background, it is noted that microvascular endothelial cells (EC) are thought to be the primary targets of acute allogeneic rejection reactions in vascularized allografts (1-5: numbers in parenthesis appear throughout the specification and correspond to publications listed numerically at the end of the description provided herein). Experiments in mice have shown that such reactions are mediated by alloantigen-specific T cells (reviewed in (6)). Consequently, many new therapeutic strategies are based on the concept of disrupting interactions between host T cells and graft EC. However, many of the molecules that mediate such interactions are sufficiently different between humans and rodents that it has been necessary to use primates even for the initial screening of inhibitory reagents. An in vivo model of human T cell-EC interactions would greatly advance the development of such reagents.
The human peripheral blood lymphocyte--severe combined immunodeficient (hu-PBL-SCID) mouse was previously introduced as a small animal model for studying the human immune system in vivo (7). C.B-17 mice homozygous for the severe combined immunodeficiency (SCID) mutation are extremely inefficient at re-arranging T and B cell antigen receptor genes (8-10)and although NK cell function is intact, these T and B cell-deficient animals are unable to mediate rejection of human xenogeneic grafts. Upon introduction of human peripheral blood lymphocytes such mice can respond to antigen by synthesizing human antibodies (7, 11-18). However, very few human T lymphocytes appear to circulate in the peripheral blood of hu-PBL-SCID mice, and there is little evidence T cell-mediated inflammatory reactions can develop in these animals (19-22). Indeed some investigators have found that human T cells become anergic in the SCID mouse microenvironment and are incapable of mediating inflammation (23).
The present invention was developed based on the belief that a major part of the difficulty in reconstituting T cell inflammatory function in the hu-PBL-SCID mouse may arise because murine EC lack critical human cell surface proteins necessary for appropriate recruitment and activation of human T lymphocytes. To explore this hypothesis, human skin with an intact dermal microvascular plexus was grafted onto a SCID mouse prior to reconstituting the mouse with human peripheral blood mononuclear leukocytes allogeneic to the skin donor. As a result it was discovered that the non-primate animal model of this invention is useful for studying human allograft rejection of donor skin as well as a model for studying immune responses in reaction to other types of foreign agents. The model generates a reproducible, stereotypic reaction in which human T cells infiltrate and destroy the human dermal microvessels.
In the present invention, the novel animal model and method of its preparation introduce several improvements over other known models. The animal model of the present invention (a) leads to perfusion of skin graft microvessels lined by human endothelial cells, and (b) produces sustained circulating levels of human T lymphocytes. In preferred embodiments, the organ graft is a split thickness adult skin specimen, rather than neonatal foreskin, for greater retention of the human microvessels (27). In addition, pretreatment of the animal model with a NK cell depletion agent in conjunction with a human lymphocyte inoculum leads to higher levels of circulating human T lymphocytes.
Although Kawamura et al have previously described "chronic" skin rejection in a SCID mouse model (20), they did not observe acute inflammation at the graft site. They only observed gradual fibrosis and some lymphocyte infiltrates, only if presensitized donors were used. Thus, their model is not appropriate for studying acute graft rejection. The animal model of the present invention differs from that of Kawamura et al in several respects. The present model demonstrates greater than 90% of the skin grafts are infiltrated by unsensitized allogeneic human T cells within a week of PBMC (peripheral blood mononuclear leukocytes) reconstitution. Secondly the observed destruction of human vascular elements within the skin is consistent with the time course seen in first set skin rejection in man (1). Thirdly, results obtained using the animal model of the present invention for allograft skin rejection suggest that the infiltrating T cells are activated in response to alloantigen. Differences in the modifications to the two models and methods employing them likely account for these discrepancies.